1. Field of Invention
The invention relates to aptamers, and more particularly to aptamers comprising at least two nucleobase-containing sequences, which are parallel or antiparallel to each other, and bound by Watson-Crick or homologous binding preferences.
2. Description of Related Art
Protein-nucleic acid complexes are known to play an important role in a variety of biological processes. See, e.g., Hill et al. “Fluorescence Approaches to Study of Protein-Nucleic Acid Complexation,” 278 Methods in Enzymology 390 (1997). For example, DNA-binding proteins are known to play an important role in gene regulation. Genes are typically regulated at the transcriptional level by DNA-binding proteins, which are referred to as transcription factors. Transcription factors regulate gene expression by specifically binding to a target nucleic acid sequence in promoter DNA.
Due to the biological importance of protein-nucleic acid interaction, a variety of methods for studying protein-nucleic acid binding characteristics have been proposed. See, e.g., Hill et al. and the references cited therein. See also, the inventors' prior U.S. patent application Ser. No. 09/224,505.
Aptamers can be designed to interact specifically with non-nucleic acid substances, such as proteins or other bodily substances. Aptamers can function as high affinity receptors for small molecule ligands or can tightly interact with target proteins for therapeutic or diagnostic purposes. The folding of an initially unstructured molecule around its ligand and forming a hydrogen-bond network with its ligand facilitate this binding. Marshall et al. “A biopolymer by any other name would bind as well: a comparison of the ligand-binding pockets of nucleic acids and proteins.” 5(6) Structure 729-734 (1997). These aptamers can be ligands used to screen for other molecules or they can be catalytic. Aptamers that are catalytic are considered approximate ribozymes, or aptazymes. To date, aptamers have been almost exclusively of single-stranded RNA. Aptamers can as well be designed to interact specifically with nucleic acid substances, other than to simply bind them on the basis of Watson-Crick base pairing between bases in nucleic acid sequences of opposite orientation. Such aptamers, if catalytic, may be fairly called aptazymes. Such specific action can be sought for therapeutic or diagnostic purposes.
A small number of RNA molecules are known to be active as catalysts and do not merely serve as the means by which information is moved out of the nucleus. Ribozymes can be self-cleaving or can cleave other RNA. This activity is understood to be dependent on the RNA's secondary structure, which can be dependent on factors such as base sequence and the inclusion of metallocations. In the past, there has been a large effort directed at building novel or improved ribozymes. Ribozymes have great utility in artificially controlling gene expression. Developers have sought to take advantage of the very specific charge patterns of nucleic acids, their bases and backbones and DNA's ability to form predictable secondary structure, based upon base sequence and predictable Watson-Crick base pairing. Nucleic acid's small dimensions and flexible nature make it well suited for constructing complexes capable of recognizing and specifically binding to features on other substances, such as proteins, and perhaps thereupon adopting tertiary structure.
Through SELEX-driven screening (U.S. Pat. No. 5,567,588 to Gold et al.), which depends upon binding to single-stranded nucleic acids mounted on biochips, researchers have discovered ribozymes, which are 100 or even 1000 fold more active catalytically.
Fernandez et al. “Pulling on Hair(pins),” 292 Science 653. (Apr. 27, 2001), reports data collected from a single molecule conformational change in a ribozyme. Fernandez et al. also reports that such essentially duplex nucleic acid structures undergo “all or none” discrete transitions in conformation, not the progressive pair by pair binding one would expect.
Researchers have disclosed a circular RNA that has enzymatic activity to cleave a separate RNA molecule at a cleavage site and RNA molecules capable of conferring stability to RNA in vivo through an endogenous ribozyme binding protein. See U.S. Pat. No. 5,712,128 to Been et al. and U.S. Pat. No. 5,985,620 to Sioud.
U.S. Pat. No. 5,840,867 to Toole discloses methods for making aptamers and aptamers that bind to biomolecules. These aptamers can be used to interfere with the normal biological function of the biomolecules, as a separation tool, a diagnostic or a therapeutic. The aptamers can be single chain or duplex RNA or DNA. However, this patent only discloses intramolecular or intermolecular Watson-Crick binding of the antiparallel variety.
Researchers have applied single-stranded RNA aptamers directed against Syrian golden hamster prion protein and the aptamers were able to recognize their specific target within a mixture of hundreds of different proteins contained in tissue homogenates thereby tending to validate the utility of aptamers. Korth et al. “Prion (PrPSc)-specific epitope defined by a monoclonal antibody.” Nature 390:74-77 (1997).
U.S. Pat. No. 6,207,388 to Grossman is directed to methods, compositions, kits and apparatus to identify and detect the presence or absence of target analytes. The compositions comprise an RNA molecule that can be an aptamer that binds to a target molecule. However, Grossman only teaches Watson-Crick antiparallel binding of nucleobases.
U.S. Pat. No. 5,858,774 to Malbon et al. provides a method of regulating a gene by introducing into a cell an antisense DNA construct. However, this patent does not teach using a nucleic acid to bind to a non-nucleic acid.
Aptamers have been used to identify and evaluate new substances, or drugs, that have a specific binding activity, or that predictably alter the binding characteristics of other binding pairs/complexes. For example, researchers have found a single-stranded DNA aptamer that binds the active site of thrombin, (a protein involved in blood coagulation), and exhibits anti-coagulation effects in vivo. Davis, “Kinetic characterization of Thrombin-Aptamer interactions.” Pharmacia Biosensor Application Note 305, 1994.
Despite the foregoing developments, there is still room in the art for aptamers of novel design with unique binding properties, and for novel uses of such aptamers.